2.3. Cooperación de un tercero
2.3.1. Consentimiento médico
between mesendodermal cells, and since it is also needed for the spreading and movement of these cells during gastrulation in vivo (Montero et al., 2005), E-cadherin could act as a downstream target of slb/wnt11 function in the regulation of cell adhesion
and migration during gastrulation. To address if Wnt11 controls the adhesion between mesendodermal progenitors by regulating the expression or subcellular distribution of E- cadherin within the early germ-ring, embryos were probed with an antibody directed against the extracellular domain of zebrafish E-cadherin (Babb and Marrs, 2004).
In wild-type embryos, E-cadherin staining could be predominantly observed at the plasma membrane and in vesicular structures in the cytosol; significantly less cytoplasmic staining was observed in slb mutant embryos (figure 14 A-C; table 06). In addition, the membrane staining appeared to be slightly weaker in wt embryos compared to slb embryos. However, the quantification of membrane intensity is still ongoing and therefore no definitive statement about E-cadherin membrane localization downstream of slb/wnt11 function can be made here.
Figure 14. Expression and intracellular localization of E-cadherin within the germ ring at the onset of
gastrulation. Confocal sections of shield stage epiblast cells at the dorsal region of the germ ring were chosen for analysis (see Methods for more details). (A,B) Face-on views of epiblast cells at shield stage (60% epiboly) stained with an antibody directed zebrafish E-cadherin in wild-type (A) and slb mutant embryos (B). The inlets show magnifications of single cells with E-cadherin positive vesicles circled. (C)
Quantification of the number of E-cadherin-positive cytoplasmic vesicles in wildtype (wt), wildtype injected with 125 pg slb/wnt11 mRNA and slb mutant epiblast cells. For each of the cases, n = 60 cells from 5 embryos were quantified; see also table 6. Scale bar in (B) = 5 µm (approximate value).
dots/cell s. d. wt + wnt11 wt slb wt + wnt11 23 3 1.00 1,95·10-2 3.27·10-8 wt 19 3 1,95·10-2 1.00 3.11·10-6 slb 10 2 3.27·10-8 3.11·10-6 1.00
Table 06. E-cadherin positive cytoplasmic ‘dots’ per cell in slb mutant embryos, wt embryos and wt
embryos injected with 125 pg slb/wnt11 mRNA. The three right-most columns display p-values, obtained by an unpaired Student’s t-test, to show that the distribution of dots is significantly changed upon slb/wnt11 function. s. d., standard deviation. For a graphical representation of the data, see figure 14.
A similar effect could be observed in slb/wnt11 mutant embryos carrying a wnt11-HA transgene under the control of a heat shock promoter, that had been heat shocked for 20 minutes at 39 ºC and fixed 30 minutes later - which is the time when wnt11-HA expression could be first detected on a western blot (Vinzenz Link, personal communication). These embryos could be rescued by heat-induced overexpression of slb/wnt11 (see table 07). After the heat shock, transgenic embryos showed an increased cytoplasmic E-cadherin staining with frequent disruptions of plasma membrane stainings. In contrast, similarly treated embryos with the same genetic background that did not carry a wnt11-HA transgene, had less E-cadherin staining in the cytoplasm and a seemingly more stable staining at the plasma membrane (figure 15 D,E). Furthermore, the differences in E-cadherin cytoplasmic distribution were not accompanied by recognizable changes in the overall expression levels of E-cadherin as determined on Western blots of early gastrula stage embryos (figure 15 A). As a control for general plasma membrane turnover downstream of slb/wnt11 function, the five-pass transmembrane protein Strabismus, C-terminally fused to an HA tag ('stbm-HA'; Jessen et al., 2002), was overexpressed in wild type embryos, injected with 125 pg slb/wnt11 mRNA, and slb mutant embryos. Embryos were fixed at shield stage and subsequently stained with an α- HA antibody, but there was no upregulation of cytoplasmic staining in wildtype embryos
compared with slb mutants3
. This staining behaviour could be consistently seen in all experiments carried out with Stbm-HA (S. Witzel, personal communication), although a definitive statement still awaits quantification. The results obtained from this experiment suggest that the enhanced cytoplasmic staining in wt embryos is specific for E-cadherin and not just the result of a generally enhanced membrane turnover (figure 15 B,C).
Taken together, these results indicate that slb/wnt11 function specifically enhances E- cadherin staining in the cytoplasm, while it downregulates E-cadherin at the plasma membrane. The expression levels of E-cadherin in the whole embryo are not affected.
Figure 15. (A) Western blot analysis of E-cadherin expression in wildtype and slb mutant embryos at the
onset of gastrulation. An Actin antibody was used as a loading control. (B-E) Confocal sections of shield
3 However, it should be noted that stbm-HA cytoplasmic staining sometimes appeared slightly increased in
stage epiblast cells at the dorsal region of the germ ring were chosen for analysis (see Methods for more details). (B,C) Face-on views of epiblast cells stained with an antibody directed against HA in a wildtype embryo injected with 125pg slb/wnt11 mRNA (B) and a slb mutant embryo (C). Both embryos were over- expressing 50pg stbm-HA. (D,E) Face-on views of epiblast cells stained with an antibody directed against zebrafish E-cadherin in a slb hs-wnt11-HA transgenic embryo and a slb non-transgenic control embryo 30’ after heat-shock. Scale bars in (C) = 10 µm and (E) = 5 µm (approximate values).